Tuberculosis (TB) is a major cause of mortality throughout the world, particularly in developing countries. There are about 8 to 9 million new cases of clinical disease reported every year and the number of deaths is estimated to be about 3 million. In the U.S. the trend of steady decline in TB has reversed and the problem is compounded by increasing numbers of drug-resistant strains. The tuberculosis complex is a group of four mycobacterial species that are genetically closely related. The three most important members are Mycobacterium tuberculosis, the major cause of human TB; Mycobacterium africanum, a major human pathogen in some populations; and Mycobacterium bovis, the cause of bovine TB. None of these mycobacteria is restricted in being pathogenic for a single host species.
In addition to being an important human disease, TB is also a major veterinary problem in many countries. Infection of cattle with M. bovis results in bovine TB and all animals showing any signs of infection are systematically slaughtered. The economic losses are thus extensive, and furthermore, cattle can serve as a reservoir for human disease.
In a majority of cases of infection, inhaled tubercle bacilli are ingested by phagocytic alveolar macrophages and are either killed or grow intracellularly to a limited extent in local lesions called tubercules. In this way the infection is limited and the primary sites of infection are walled off without any symptoms of disease being observed. Such individuals have a lifetime risk of about 10% for developing active disease. In a latter eventuality, bacilli spread from the site of infection in the lung, through the lung and via lymphatics or blood to other parts of the body producing characteristic solid caseous (cheese-like) necrosis in which bacilli survive. If the necrotic reaction expands breaking into a bronchus, or in the worst case, if the solid necrosis liquefy, a rapid proliferation of the bacilli occurs. The pathological and inflammatory processes set in motion then produce the characteristic weakness, fever, chest pain, cough and bloody sputum which are the hallmarks of active TB.
Effective treatment of TB with antibiotics exists. However, this is expensive and requires prolonged administration of a combination of drugs. There is a problem in compliance with the drug administration regime because of the extended time periods involved and this has contributed to the appearance of drug-resistant strains. There is a recognized vaccine for TB which is an attenuated form of M. bovis, known as BCG (bacille Calmette Guerin). This strain was developed in 1921 and the basis for its attenuation is still not known (ref. 1--throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure). The efficacy of BCG as a TB vaccine is a subject of controversy and has been estimated in various trials to be anywhere between 0 and 70%.
The molecular basis for the virulence and pathogenesis of M. tuberculosis has not been extensively described. Some virulence factors, particularly those related to the sigma factors have been recently identified (ref. 2). M. tuberculosis can enter non-phagocytic cells in culture, such as HeLa cells (ref. 3) and once inside can multiply and survive. Recently, a protein encoded by a DNA fragment (1535 bp long) from a strain of M. tuberculosis (H37Ra) was reported to mediate the entry of the bacterium and its survival in mammalian cells (ref. 4). This DNA fragment when introduced into a non-pathogenic strain of E. coli is able to confer invasiveness to the bacterium, and survival for up to 24 hours in human macrophages. The mce (mycobacterial cell entry) gene was mapped to an Open Reading Frame (ORF) extending from position 182 to 810 on the 1535 bp DNA fragment mentioned above and encodes a protein of molecular weight between 22 and 27 kDa. Subsequent work has shown the gene described in ref. 4 is not a full length gene.
In copending U.S. patent application Ser. No. 08/677,970 filed Jul. 10, 1996, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference, there is described the isolation and characterization of genes encoding proteins of mycobacteria associated with cell binding and cell entry and the protein encoded thereby. This gene is referred to herein as the Mycobacterial cell entry (mce) gene and the encoded protein the Mycobacterial cell entry protein (Mcep).
Mycobacterial infection may lead to serious disease. It would be advantageous to provide attenuated strains of Mycobacterium wherein the mycobacterial cell entry gene is disabled, and immunogenic preparations including vaccines comprising the same.